摘要
目的 建立稳定表达血管内皮生长因子的人脐静脉内皮细胞系 ,研究转染后对内皮细胞生长和一氧化氮分泌的影响。方法 将真核表达载体PCD2 VEGF12 1用阳离子脂质体介导 ,转染人脐静脉内皮细胞系细胞 ,分别用RT PCR、免疫组化、Miles实验检测血管内皮生长因子的转录、蛋白质的表达及其生物学活性 ,检测转染后内皮细胞生长状况和培养基中一氧化氮水平。结果 RT PCR检测出了转录血管内皮生长因子的稳定转染细胞克隆 ,该单克隆细胞的免疫组化检测血管内皮生长因子蛋白质表达结果呈阳性 ,Miles实验表明其表达产物具有生物学活性 ,而作为对照的转空白质粒细胞和未转染细胞上述实验结果皆为阴性 ;转染后内皮细胞生长明显加快 ,一氧化氮水平在 4 8h和 72h时明显增高。结论 成功建立了稳定表达血管内皮生长因子的内皮细胞系 ,血管内皮生长因子转染可促进内皮细胞生长和一氧化氮分泌。
Objective To establish on growth of endothelial cells and secretion of nitric oxide.Methods The eukaryotic expression vector PCD 2 VEGF 121 were transfected into HUVEC line cells mediated by lipofect AMINE.The positive clones were obtained by the screen of G 418 .The transcription and expression of VEGF gene were investigated by RT PCR and immunocytochenistry respectively.The experiment of Miles wes applied for the assay of the biological activity of the protein of the VEGF produced by the HUVEC line cells which transfected PCD 2 CEGF 121 .The growth curve was made for comparison with that of non transfected HUVEC line cells.The level of nitric oxide was detected by nitric oxide kit.Results A positive clone cells from which transcripted the MRNA of VEGF 121 gene was obtained by RT PCR;The positive results of the immunocytochemistry were found and the highly biological activity of VEGF in the media was detected each only in the positive clone cells.The doublingtime of the positive colon cells was shorter than that of the non transfected HUVEC line cells calculated from the growth curve.The level of nitric oxide increased in transfected endothelial cells.Conclusion The HUVEC line monoclonal cells with the stable expression of VEGF gene has been established successfully.VEGF may promote growth of endothelial cells and secretion of nitric oxide.
出处
《重庆医学》
CAS
CSCD
2003年第1期20-22,共3页
Chongqing medicine
基金
湖南省教委重点课题项目资助 (972 0 0 9)
